Abstract
BackgroundSoybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes.ResultsWe report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development.ConclusionThe nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.
Highlights
Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance
Scoring resistance using a hairy root/nematode demographics assay In soybean, Medicago truncatula and other plants for which stable transformation is difficult, transgenic "hairy roots" generated through A. rhizogenes-mediated transformation are often used to investigate gene function
Cyst formation is typically counted after 4 weeks but because the root tissues used often exhibit significantly reduced vigor 3-4 weeks into such assays, and to expedite and possibly improve resistance scoring, we investigated the scoring of resistance phenotypes at an earlier time point
Summary
Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S The Rhg locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. Soybean cyst nematode (SCN, Heterodera glycines) is an obligate, sedentary endoparasite that is consistently the most damaging pest of soybean in the U.S [1]. After the first molt within the egg, SCN second stage juveniles (J2) hatch, move through the soil, penetrate roots and move female body develops into a hardened cyst that encases the eggs. Soybean cyst nematodes infect and grow in the roots of both resistant and susceptible cultivars [2,5]. The available SCN resistance in soybean is partial, and can be observed as a reduced number of females that develop compared to the number that develop on inoculated susceptible cultivar controls [6]. Soybean cultivars are generally classified as strongly resistant to SCN if the FI is less than 10%; partial levels of resistance can be useful [8]
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